Leucaena sp. recombinant cinnamyl alcohol dehydrogenase: Purification and physicochemical characterization

Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LlCAD2) isolated from Leucaena leucocephala. LlCAD2 is 80 kDa homo-dimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with Kcat/Km of 11.6 × 106 (M−1 s−1), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 °C, with the deactivation rate constant (Kd*) and half-life (t1/2) of 0.002 and 5 h, respectively. LlCAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (Kd*) and half-life (t1/2) of 7.5 × 10−4 and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn2+ per dimer, however, was inhibited in presence of externally supplemented Zn2+ ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents.